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Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1.

Sacchetti P, Carpentier R, Ségard P, Olivé-Cren C, Lefebvre P - Nucleic Acids Res. (2006)

Bottom Line: Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase.We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity.Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

View Article: PubMed Central - PubMed

Affiliation: INSERM U459, Faculté de Médecine de Lille, 59045 Lille, France. paola.sacchetti@ki.se

ABSTRACT
The orphan nuclear receptor nurr1 (NR4A2) is an essential transcription factor for the acquisition and maintenance of the phenotype of dopamine (DA)-synthesizing neurons in the mesencephalon. Although structurally related to ligand-regulated nuclear receptors, nurr1 is functionally atypical due to its inability to bind a cognate ligand and to activate transcription following canonical nuclear receptor (NR) rules. Importantly, the physiological stimuli that activate this NR and the signaling proteins that regulate its transcriptional activity in mesencephalic neurons are unknown. We used an affinity chromatography approach and CSM14.1 cells of mesencephalic origin to isolate and identify several proteins that interact directly with nurr1 and regulate its transcriptional activity. Notably, we demonstrate that the mitogen-activated protein kinases, ERK2 and ERK5, elevate, whereas LIM Kinase 1 inhibits nurr1 transcriptional activity. Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase. We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity. Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

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Nurr1 is phosphorylated by BMK1/ERK5 MAP kinase. (A) DNA immunoprecipitation of nurr1 and endogenous ERK5 recruited to NBREs in cells overexpressing HA-nurr1, but not to a promoter lacking nurr1 response elements, pGL3-tk-Luc. (B) Endogenous ERK5 immunoprecipitated from untransfected CSM14.1 cells was incubated with GST-A/BΔ nurr1 or GST fusion proteins and [γ-32P]ATP for 30 min. Arrows indicate phosphorylated nurr1. For activation of MEK-ERK5 pathway, cells were stimulated for 15 min with 100 ng/ml of EGF. (C) Analysis of luciferase activity induced by coexpression of wt HA-ERK5 and wt pCMX-nurr1 or point mutations S89A, T168A and S177A on NBRE3xtkLuc. Normalized luciferase activity of wt nurr1 plus ERK5 was set to 100% and all other values were compared to it. Data are the means of ± S.E. (bars) of five independent experiments (n = 3 in each experiment).
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fig6: Nurr1 is phosphorylated by BMK1/ERK5 MAP kinase. (A) DNA immunoprecipitation of nurr1 and endogenous ERK5 recruited to NBREs in cells overexpressing HA-nurr1, but not to a promoter lacking nurr1 response elements, pGL3-tk-Luc. (B) Endogenous ERK5 immunoprecipitated from untransfected CSM14.1 cells was incubated with GST-A/BΔ nurr1 or GST fusion proteins and [γ-32P]ATP for 30 min. Arrows indicate phosphorylated nurr1. For activation of MEK-ERK5 pathway, cells were stimulated for 15 min with 100 ng/ml of EGF. (C) Analysis of luciferase activity induced by coexpression of wt HA-ERK5 and wt pCMX-nurr1 or point mutations S89A, T168A and S177A on NBRE3xtkLuc. Normalized luciferase activity of wt nurr1 plus ERK5 was set to 100% and all other values were compared to it. Data are the means of ± S.E. (bars) of five independent experiments (n = 3 in each experiment).

Mentions: To clarify the mechanism by which ERK5 transduces its activating properties on nurr1, we asked if the transcriptional effects observed were correlated with an interaction between nurr1 and ERK5 on DNA. Because of the lack of direct nurr1 binding response elements in the promoters of DAergic target genes, we used the NBRE reporter construct for our DNA immunoprecipitation assays, a system previously used to explore NRs transactivation mechanisms (27). Thus, we tested the recruitment of the two proteins on the NBRE-containing reporter plasmid by DNA immunoprecipitation assay (Figure 6A) using antibodies specific to nurr1 and ERK5. Immunoprecipitation followed by PCR amplification of the DNA fragment encompassing the three NBRE response elements showed that nurr1 and ERK5 were both strongly bound to this response element in vivo, suggesting that ERK5 interacts with DNA-bound nurr1 and acts as transcriptional activator. This recruitment was not observed on a promoter deprived of the nurr1 binding sites (pGL3-tk-Luc; Figure 6A). Identical DNA immunoprecipitation results were obtained in CSM14.1 and PC12 cells, confirming our functional studies obtained in the later cell type (Figure 5). These studies suggested that the kinase activity of ERK5 was involved in enhancing nurr1 activity. Thus, we wondered if nurr1 could be a new substrate for this kinase and tested the ability of ERK5 to phosphorylate GST–nurr1 fusion proteins in vitro. Therefore, we immunoprecipitated endogenous ERK5 from CSM14.1 cells and co-incubated GST fusion proteins coding for the A/B domain of nurr1 with the precipitated kinase in the presence of radioactive ATP. As shown in Figure 6B, nurr1 A/B domain was significantly phosphorylated by ERK5, while the GST protein alone was not a substrate for this kinase. We attempted to activate the endogenous MEK-ERK5 pathway by stimulating CSM14.1 cells with EGF for 15 min and verify the phosphorylation state of nurr1. Figure 6B shows the EGF-induced phosphorylation of ERK5, as shown by western blot, however no changes were observed in the phosphorylation state of nurr1. These data would suggest once again that the basal activity of ERK5 is responsible for the phosphorylation of nurr1. To establish which amino acids were implicated in ERK5-mediated phosphorylation, several residues in the A/B domain of nurr1 were mutated to alanines. First, we observed that all point mutations had a high basal activity which was comparable to wt nurr1 plus ERK5 (Figure 6C) and that, similarly to wt nurr1, the S89A mutant response was significantly enhanced in the presence of ERK5. On the other hand, the activity of the T168A mutant was only slightly increased in the presence of the kinase, while the S177A mutant lost completely its capacity to be activated by ERK5. Thus, residues T168 and S177 appeared to be implicated in the activation of nurr1 transcriptional activity by ERK5. All together these results suggest that nurr1 is a downstream target of the MEK5-ERK5 signaling pathway, as suggested by the recruitment of ERK5 to the NBRE, the phosphorylation of nurr1 N-terminal by this same kinase and the capacity of MEK5 and ERK5 to increase nurr1 transcriptional activity.


Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1.

Sacchetti P, Carpentier R, Ségard P, Olivé-Cren C, Lefebvre P - Nucleic Acids Res. (2006)

Nurr1 is phosphorylated by BMK1/ERK5 MAP kinase. (A) DNA immunoprecipitation of nurr1 and endogenous ERK5 recruited to NBREs in cells overexpressing HA-nurr1, but not to a promoter lacking nurr1 response elements, pGL3-tk-Luc. (B) Endogenous ERK5 immunoprecipitated from untransfected CSM14.1 cells was incubated with GST-A/BΔ nurr1 or GST fusion proteins and [γ-32P]ATP for 30 min. Arrows indicate phosphorylated nurr1. For activation of MEK-ERK5 pathway, cells were stimulated for 15 min with 100 ng/ml of EGF. (C) Analysis of luciferase activity induced by coexpression of wt HA-ERK5 and wt pCMX-nurr1 or point mutations S89A, T168A and S177A on NBRE3xtkLuc. Normalized luciferase activity of wt nurr1 plus ERK5 was set to 100% and all other values were compared to it. Data are the means of ± S.E. (bars) of five independent experiments (n = 3 in each experiment).
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fig6: Nurr1 is phosphorylated by BMK1/ERK5 MAP kinase. (A) DNA immunoprecipitation of nurr1 and endogenous ERK5 recruited to NBREs in cells overexpressing HA-nurr1, but not to a promoter lacking nurr1 response elements, pGL3-tk-Luc. (B) Endogenous ERK5 immunoprecipitated from untransfected CSM14.1 cells was incubated with GST-A/BΔ nurr1 or GST fusion proteins and [γ-32P]ATP for 30 min. Arrows indicate phosphorylated nurr1. For activation of MEK-ERK5 pathway, cells were stimulated for 15 min with 100 ng/ml of EGF. (C) Analysis of luciferase activity induced by coexpression of wt HA-ERK5 and wt pCMX-nurr1 or point mutations S89A, T168A and S177A on NBRE3xtkLuc. Normalized luciferase activity of wt nurr1 plus ERK5 was set to 100% and all other values were compared to it. Data are the means of ± S.E. (bars) of five independent experiments (n = 3 in each experiment).
Mentions: To clarify the mechanism by which ERK5 transduces its activating properties on nurr1, we asked if the transcriptional effects observed were correlated with an interaction between nurr1 and ERK5 on DNA. Because of the lack of direct nurr1 binding response elements in the promoters of DAergic target genes, we used the NBRE reporter construct for our DNA immunoprecipitation assays, a system previously used to explore NRs transactivation mechanisms (27). Thus, we tested the recruitment of the two proteins on the NBRE-containing reporter plasmid by DNA immunoprecipitation assay (Figure 6A) using antibodies specific to nurr1 and ERK5. Immunoprecipitation followed by PCR amplification of the DNA fragment encompassing the three NBRE response elements showed that nurr1 and ERK5 were both strongly bound to this response element in vivo, suggesting that ERK5 interacts with DNA-bound nurr1 and acts as transcriptional activator. This recruitment was not observed on a promoter deprived of the nurr1 binding sites (pGL3-tk-Luc; Figure 6A). Identical DNA immunoprecipitation results were obtained in CSM14.1 and PC12 cells, confirming our functional studies obtained in the later cell type (Figure 5). These studies suggested that the kinase activity of ERK5 was involved in enhancing nurr1 activity. Thus, we wondered if nurr1 could be a new substrate for this kinase and tested the ability of ERK5 to phosphorylate GST–nurr1 fusion proteins in vitro. Therefore, we immunoprecipitated endogenous ERK5 from CSM14.1 cells and co-incubated GST fusion proteins coding for the A/B domain of nurr1 with the precipitated kinase in the presence of radioactive ATP. As shown in Figure 6B, nurr1 A/B domain was significantly phosphorylated by ERK5, while the GST protein alone was not a substrate for this kinase. We attempted to activate the endogenous MEK-ERK5 pathway by stimulating CSM14.1 cells with EGF for 15 min and verify the phosphorylation state of nurr1. Figure 6B shows the EGF-induced phosphorylation of ERK5, as shown by western blot, however no changes were observed in the phosphorylation state of nurr1. These data would suggest once again that the basal activity of ERK5 is responsible for the phosphorylation of nurr1. To establish which amino acids were implicated in ERK5-mediated phosphorylation, several residues in the A/B domain of nurr1 were mutated to alanines. First, we observed that all point mutations had a high basal activity which was comparable to wt nurr1 plus ERK5 (Figure 6C) and that, similarly to wt nurr1, the S89A mutant response was significantly enhanced in the presence of ERK5. On the other hand, the activity of the T168A mutant was only slightly increased in the presence of the kinase, while the S177A mutant lost completely its capacity to be activated by ERK5. Thus, residues T168 and S177 appeared to be implicated in the activation of nurr1 transcriptional activity by ERK5. All together these results suggest that nurr1 is a downstream target of the MEK5-ERK5 signaling pathway, as suggested by the recruitment of ERK5 to the NBRE, the phosphorylation of nurr1 N-terminal by this same kinase and the capacity of MEK5 and ERK5 to increase nurr1 transcriptional activity.

Bottom Line: Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase.We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity.Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

View Article: PubMed Central - PubMed

Affiliation: INSERM U459, Faculté de Médecine de Lille, 59045 Lille, France. paola.sacchetti@ki.se

ABSTRACT
The orphan nuclear receptor nurr1 (NR4A2) is an essential transcription factor for the acquisition and maintenance of the phenotype of dopamine (DA)-synthesizing neurons in the mesencephalon. Although structurally related to ligand-regulated nuclear receptors, nurr1 is functionally atypical due to its inability to bind a cognate ligand and to activate transcription following canonical nuclear receptor (NR) rules. Importantly, the physiological stimuli that activate this NR and the signaling proteins that regulate its transcriptional activity in mesencephalic neurons are unknown. We used an affinity chromatography approach and CSM14.1 cells of mesencephalic origin to isolate and identify several proteins that interact directly with nurr1 and regulate its transcriptional activity. Notably, we demonstrate that the mitogen-activated protein kinases, ERK2 and ERK5, elevate, whereas LIM Kinase 1 inhibits nurr1 transcriptional activity. Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase. We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity. Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

Show MeSH